Hybrid compounds generated by the introduction of a nogalamycin-producing plasmid into Streptomyces argillaceus † Tero Kunnari,* a Karel D. Klika, b Gloria Blanco, c Carmen Méndez, c Pekka Mäntsälä, d Juha Hakala,‡ a Reijo Sillanpää, e Petri Tähtinen, b Jose Salas c and Kristiina Ylihonko a a Galilaeus Oy, P.O. Box 113, FIN-20781 Kaarina, Finland. E-mail: tero.kunnari@galilaeus.fi; Fax: +358-2-2731460; Tel: +358-2-2741450 b Structural Chemistry Group, Department of Chemistry, University of Turku, Vatselankatu 2, FIN-20014 Turku, Finland c Departamento de Biologia Funcional e Instituto, Universitario de Biotecnologia de Asturias, ESP-33006 Oviedo, Spain d Department of Biochemistry, University of Turku, Vatselankatu 2, FIN-20014 Turku, Finland e Department of Chemistry, University of Jyväskylä, Survontie 9, FIN-40500 Jyväskylä, Finland Received (in Cambridge, UK) 11th January 2002, Accepted 22nd May 2002 First published as an Advance Article on the web 11th July 2002 The combination of genetic material from different antibiotic-producing organisms is a versatile and ever-expanding approach for the production of novel, hybrid compounds possessing bioactivity. The introduction of a plasmid (pSY21b) derived from Streptomyces nogalater and encoding PKS for nogalamycin production into the host strain S. argillaceus A43 led to the production of three new compounds in addition to the normally produced mithramycin. The new compounds are hybrids in the sense that they share features associated with the genes of both the host and the introduced plasmid. The structural elucidation of the novel compounds relied primarily on NMR spectroscopy, which revealed the three hybrids to be glycosides with the same aglycone common to all. Determination of the relative stereochemistry within the aglycone unit was confirmed by single-crystal X-ray analysis of the aglycone, which also revealed the tautomeric equilibrium to be in a very different position in comparison to that in the solution state. The glycosylation profile was clearly determined by the host, as the typical mithramycin sugars, -oliose, -olivose, and -mycarose, were all expressed. Notable for the mutant was the high titre of the shunt products, 60% of the metabolic output, together with a lack of structural diversity of the hybrid aglycone present in the products, two features which are not normally observed. Introduction Since its inception 1 almost two decades ago, the combination of genetic material from different antibiotic-producing organisms has proved to be a versatile, and ever-expanding approach 2 for the production of novel, hybrid compounds possessing latent bioactivity with the potential of being incorporated into the drug discovery process as drug candidates. Considerable attention has been directed towards highlighting the benefits and advantages of both the genetic engineering of antibiotic-producing microbes 3–5 and bio- transformation using whole-cell techniques 5,6 and the realisation of the practical application of this approach as a routine, predictable methodology appears imminent. Mithramycin (1) (also known as aureolic acid, plicamycin, mithracin ® , etc. Fig. 1) is a bis(oligosaccharide) antibiotic produced by Streptomyces argillaceus and some other Streptomyces strains, 7 whose structural elucidation, 8 since its discovery in the 1950s, 9 has been contraversial. It is a highly toxic antibiotic possessing both antineoplastic and hypo- calcemic properties. 10,11 The antibiotics UCH9, the chromo- cyclomycins, the chromomycins, and the olivomycins are all structurally and biochemically closely related to mithramycin (1) and, together, they are classified within the aureolic acid † Electronic supplementary information (ESI) available: edited GAUSSIAN output of DFT calculations and HyperChem file of the calculated structure. See http://www.rsc.org/suppdata/p1/b2/b200444p/ ‡ Current address: Lividans Oy, Tykistökatu 4 D, FIN-20520 Turku, Finland. group of compounds. 8 They are potent inhibitors of in vitro and in vivo RNA synthesis as a consequence of their ability to form stable complexes with DNA. 12 However, unlike intercalating dyes and antibiotics, mithramycin (1) does not induce uncoiling of DNA. Despite their demonstrated potency in chemotherapy, the only aureolic acid that is presently in clinical use, albeit limited, is mithramycin (1). Within this context mithramycin (1) holds promise as a structural template for the application of hybrid techniques to generate novel compounds and thus potential new drug candidates. The structure was initially, and correctly, elucidated using chemical degradation and derivitisation techniques. 13 Subsequently, it was reinvestigated by several workers resulting in the publication of various erroneous structures, 13–15 mainly with respect to the identity, anomeric state, or linkage position of the attendant sugar units. These incorrect structures, needless to say, underpinned a poor appreciation of the mithramycin (1) biosynthetic pathway and now that modern NMR methods have firmly established the correct structure of mithramycin 8 (1), a better comprehension of the biosynthesis of mithramycin (1) is possible. Nogalamycin (2) (Fig. 1) is an antitumour antibiotic belonging to the anthracycline group of compounds. Anthra- cyclines are useful cytotoxic compounds and some of them, e.g. doxorubicin and several analogues, are used clinically for the treatment of various malignancies. Like mithramycin (1), nogalamycin (2) is also biosynthesised via a polyketide pathway and is thus classified as an aromatic type II polyketide on the basis of the molecular genetics of the anthracyclines. The biosynthetic pathways of both mithramycin 16 (1) and nogalamycin 17 (2) are today well understood regarding the 1 PERKIN 1818 J. Chem. Soc., Perkin Trans. 1, 2002, 1818–1825 DOI: 10.1039/b200444p This journal is © The Royal Society of Chemistry 2002